US11330776B2ActiveUtilityPatentIndex 79

Methods and compositions for rapid plant transformation

Assignee: PIONEER HI BRED INTPriority: Oct 30, 2015Filed: Aug 26, 2016Granted: May 17, 2022

Est. expiryOct 30, 2035(~9.3 yrs left)· nominal 20-yr term from priority

Inventors:ANAND AJITH ARLING MAREN L DA SILVA CONCEICAO ALEXANDRE GORDON-KAMM WILLIAM JAMES HASTINGS CRAIG E HOERSTER GEORGE J JONES TODD J KLEIN THEODORE MITCHELL LA ROTA CARLOS M LOWE KEITH S TIWARI SHIV BAHADUR WANG NING WU XINLI EMILY

C07K 14/415A01H 4/008C12N 15/8216C12N 15/821C12N 15/8201C12N 15/8213Y02A40/146

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Claims

Abstract

The disclosure pertains to methods and compositions for the rapid and efficient transformation of plants. The disclosure further provides methods for producing a transgenic plant, comprising (a) transforming a cell of an explant with an expression construct comprising (i) a nucleotide sequence encoding a WUS/WOX homeobox polypeptide; (ii) a nucleotide sequence encoding a polypeptide comprising two AP2-DNA binding domains; or (iii) a combination of (i) and (ii); and (b) allowing expression of the polypeptide of (a) in each transformed cell to form a regenerable plant structure in the absence of exogenous cytokinin, wherein no callus is formed; and (c) germinating the regenerable plant structure to form the transgenic plant. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present disclosure.

Claims

exact text as granted — not AI-modified

That which is claimed: 
     
       1. A method for producing a transgenic Poaceae plant, comprising:
 (a) transforming a cell of a Poaceae explant with an expression construct comprising a heterologous gene of interest; and
 (i) a nucleotide sequence encoding a WUS/WOX homeobox polypeptide; and 
 (ii) a nucleotide sequence encoding a Babyboom (BBM) polypeptide or an Ovule Development Protein 2 (ODP2) polypeptide; and 
 
 (b) allowing expression of the expression construct of (a) in each transformed cell for about 0 to about 7 days or for about 0 to about 14 days after initiation of transforming the cell, wherein a somatic embryo is formed within about 0 to about 7 days or within about 0 to about 14 days after initiation of transforming the cell in the absence of exogenous cytokinin; and 
 (c) germinating the somatic embryo to form the transgenic Poaceae plant. 
 
     
     
       2. The method of  claim 1 , wherein germinating comprises transferring the somatic embryo to a maturation medium and forming the transgenic Poaceae plant. 
     
     
       3. The method of  claim 1 , wherein the expression construct further comprises a nucleotide sequence encoding a site-specific recombinase selected from FLP, Cre, SSV1, lambda Int, phi C31 Int, HK022, R, Gin, Tn1721, CinH, ParA, Tn5053, Bxb1, TP907-1, or U153. 
     
     
       4. The method of  claim 3 , wherein the nucleotide sequence encoding a site-specific recombinase is operably linked to a constitutive promoter, an inducible promoter, or a developmentally regulated promoter. 
     
     
       5. The method of  claim 1 , wherein (c) is performed in the presence of exogenous cytokinin. 
     
     
       6. The method of  claim 1 , wherein the nucleotide sequence encoding the WUS/WOX homeobox polypeptide and the nucleotide sequence encoding the BBM polypeptide or the ODP2 polypeptide is operably linked to a promoter selected from an inducible promoter, a developmentally regulated promoter, or a constitutive promoter. 
     
     
       7. The method of  claim 4 , wherein the constitutive promoter is selected from UBI, LLDAV, EVCV, DMMV, BSV(AY) PRO, CYMV PRO FL, UBIZM PRO, SI-UB3 PRO, SB-UBI PRO (ALT1), USB1ZM PRO, ZM-GOS2 PRO, ZM-H1B PRO (1.2 KB), IN2-2, NOS, the −135 version of 35S, or ZM-ADF PRO (ALT2);
 the inducible promoter is selected from AXIG1, DR5, XVE, GLB1, OLE, LTP2, HSP17.7, HSP26, HSP18A, or promoters activated by tetracycline, ethamethsulfuron or chlorsulfuron; and 
 the developmentally regulated promoter is selected from PLTP, PLTP1, PLTP2, PLTP3, SDR, LGL, LEA-14A, or LEA-D34. 
 
     
     
       8. The method of  claim 6 , wherein the constitutive promoter is selected from UBI, LLDAV, EVCV, DMMV, BSV(AY) PRO, CYMV PRO FL, UBIZM PRO, SI-UB3 PRO, SB-UBI PRO (ALT1), USB1ZM PRO, ZM-GOS2 PRO, ZM-H1B PRO (1.2 KB), IN2-2, NOS, the −135 version of 35S, or ZM-ADF PRO (ALT2);
 the inducible promoter is selected from AXIG1, DR5, XVE, GLB1, OLE, LTP2, HSP17.7, HSP26, HSP18A, or promoters activated by tetracycline, ethamethsulfuron or chlorsulfuron; and 
 the developmentally regulated promoter is selected from PLTP, PLTP1, PLTP2, PLTP3, SDR, LGL, LEA-14A, or LEA-D34. 
 
     
     
       9. A method for producing a transgenic Poaceae plant, comprising:
 (a) transforming a cell of a Poaceae explant with an expression construct comprising a heterologous gene of interest; and
 (i) a nucleotide sequence encoding a WUS/WOX homeobox polypeptide; and 
 (ii) a nucleotide sequence encoding a Babyboom (BBM) polypeptide or an Ovule Development Protein 2 (ODP2) polypeptide; and 
 
 (b) allowing expression of the expression construct of (a) in each transformed cell for about 0 to about 7 days or for about 0 to about 14 days after initiation of transforming the cell, wherein a somatic embryo is formed within about 0 to about 7 days or within about 0 to about 14 days after initiation of transforming the cell in the absence of exogenous cytokinin; and 
 (c) germinating the somatic embryo to form the transgenic Poaceae plant; 
 
       wherein the WUS/WOX homeobox polypeptide comprises the amino acid sequence of any of SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16; or wherein the WUS/WOX homeobox polypeptide is encoded by the nucleotide sequence of any of SEQ ID NO: 3, 5, 7, 9, 11, 13, or 15; 
       wherein the polypeptide comprising the BBM polypeptide or the ODP2 polypeptide comprises the amino acid sequence of any of SEQ ID NO: 18, 20, 63, 65, or 67; or wherein the polypeptide comprising the BBM polypeptide or the ODP2 polypeptide is encoded by the nucleotide sequence of any of SEQ ID NO: 17, 19, 21, 62, 64, 66, or 68. 
     
     
       10. The method of  claim 9 , wherein germinating comprises transferring the somatic embryo to a maturation medium and forming the transgenic Poaceae plant. 
     
     
       11. The method of  claim 9 , wherein the expression construct further comprises a nucleotide sequence encoding a site-specific recombinase selected from FLP, Cre, SSV1, lambda Int, phi C31 Int, HK022, R, Gin, Tn1721, CinH, ParA, Tn5053, Bxb1, TP907-1, or U153. 
     
     
       12. The method of  claim 11 , wherein the nucleotide sequence encoding a site-specific recombinase is operably linked to a constitutive promoter, an inducible promoter, or a developmentally regulated promoter. 
     
     
       13. The method of  claim 9 , wherein (c) is performed in the presence of exogenous cytokinin. 
     
     
       14. The method of  claim 9 , wherein the nucleotide sequence encoding the WUS/WOX homeobox polypeptide and the nucleotide sequence encoding a polypeptide comprising the BBM polypeptide or the ODP2 polypeptide is operably linked to an inducible promoter, a developmentally regulated promoter, or a constitutive promoter. 
     
     
       15. The method of  claim 12 , wherein the constitutive promoter is selected from UBI, LLDAV, EVCV, DMMV, BSV (AY) PRO, CYMV PRO FL, UBIZM PRO, SI-UB3 PRO, SB-UBI PRO (ALT1), USB1ZM PRO, ZM-GOS2 PRO, ZM-H1B PRO (1.2 KB), IN2-2, NOS, the −135 version of 35S, or ZM-ADF PRO (ALT2);
 the inducible promoter is selected from AXIG1, DR5, XVE, GLB1, OLE, LTP2, HSP17.7, HSP26, HSP18A, or promoters activated by tetracycline, ethamethsulfuron or chlorsulfuron; and 
 the developmentally regulated promoter is selected from PLTP, PLTP1, PLTP2, PLTP3, LGL, LEA-14A, or LEA-D34. 
 
     
     
       16. The method of  claim 14 , wherein the constitutive promoter is selected from UBI, LLDAV, EVCV, DMMV, BSV(AY) PRO, CYMV PRO FL, UBIZM PRO, SI-UB3 PRO, SB-UBI PRO (ALT1), USB1ZM PRO, ZM-GOS2 PRO, ZM-H1B PRO (1.2 KB), IN2-2, NOS, the −135 version of 35S, or ZM-ADF PRO (ALT2);
 the inducible promoter is selected from AXIG1, DR5, XVE, GLB1, OLE, LTP2, HSP17.7, HSP26, HSP18A, or promoters activated by tetracycline, ethamethsulfuron or chlorsulfuron; and 
 the developmentally regulated promoter is selected from PLTP, PLTP1, PLTP2, PLTP3, SDR, LGL, LEA-14A, or LEA-D34. 
 
     
     
       17. A method for producing a transgenic Poaceae plant comprising:
 (a) transforming a cell of a Poaceae explant with an expression construct comprising a heterologous gene of interest; and
 (i) a nucleotide sequence encoding a WUS/WOX homeobox polypeptide; and 
 (ii) a nucleotide sequence encoding a Babyboom (BBM) polypeptide or an Ovule Development Protein 2 (ODP2) polypeptide; and 
 
 (b) allowing expression of the expression construct of (a) in each transformed cell for about 0 to about 7 days or for about 0 to about 14 days after initiation of transforming the cell, wherein a somatic embryo is formed within about 0 to about 7 days or within about 0 to about 14 days after initiation of transforming the cell in the absence of exogenous cytokinin; and 
 (c) germinating the somatic embryo of (b) for about 14 to about 60 days to form a plantlet; and 
 (d) allowing the plantlet of (c) to grow into a Poaceae plant. 
 
     
     
       18. The method of  claim 17 , wherein germinating comprises transferring the somatic embryo to a maturation medium and forming the transgenic Poaceae plant. 
     
     
       19. The method of  claim 17 , wherein the expression construct further comprises a nucleotide sequence encoding a site-specific recombinase selected from FLP, Cre, SSV1, lambda Int, phi C31 Int, HK022, R, Gin, Tn1721, CinH, ParA, Tn5053, Bxb1, TP907-1, or U153. 
     
     
       20. The method of  claim 19 , wherein the nucleotide sequence encoding a site-specific recombinase is operably linked to a constitutive promoter, an inducible promoter, or a developmentally regulated promoter. 
     
     
       21. The method of  claim 17 , wherein (c) is performed in the presence of exogenous cytokinin. 
     
     
       22. The method of  claim 17 , wherein the nucleotide sequence encoding the WUS/WOX homeobox polypeptide and the nucleotide sequence encoding a polypeptide comprising the BBM polypeptide or the ODP2 polypeptide is operably linked to an inducible promoter, a developmentally regulated promoter, or a constitutive promoter. 
     
     
       23. The method of  claim 20 , wherein the constitutive promoter is selected from UBI, LLDAV, EVCV, DMMV, BSV (AY) PRO, CYMV PRO FL, UBIZM PRO, SI-UB3 PRO, SB-UBI PRO (ALT1), USB1ZM PRO, ZM-GOS2 PRO, ZM-H1B PRO (1.2 KB), IN2-2, NOS, the −135 version of 35S, or ZM-ADF PRO (ALT2);
 the inducible promoter is selected from AXIG1, DR5, XVE, GLB1, OLE, LTP2, HSP17.7, HSP26, HSP18A, or promoters activated by tetracycline, ethamethsulfuron or chlorsulfuron; and 
 the developmentally regulated promoter is selected from PLTP, PLTP1, PLTP2, PLTP3, LGL, LEA-14A, or LEA-D34. 
 
     
     
       24. The method of  claim 22 , wherein the constitutive promoter is selected from UBI, LLDAV, EVCV, DMMV, BSV(AY) PRO, CYMV PRO FL, UBIZM PRO, SI-UB3 PRO, SB-UBI PRO (ALT1), USB1ZM PRO, ZM-GOS2 PRO, ZM-H1B PRO (1.2 KB), IN2-2, NOS, the −135 version of 35S, or ZM-ADF PRO (ALT2);
 the inducible promoter is selected from AXIG1, DR5, XVE, GLB1, OLE, LTP2, HSP17.7, HSP26, HSP18A, or promoters activated by tetracycline, ethamethsulfuron or chlorsulfuron; and 
 the developmentally regulated promoter is selected from PLTP, PLTP1, PLTP2, PLTP3, SDR, LGL, LEA-14A, or LEA-D34. 
 
     
     
       25. The method of  claim 1 , wherein the WUS/WOX homeobox polypeptide comprises the amino acid sequence of any of SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16, or wherein the WUS/WOX homeobox polypeptide is encoded by the nucleotide sequence of any of SEQ ID NO: 3, 5, 7, 9, 11, 13, or 15 and wherein the BBM polypeptide or the Ovule Development Protein 2 (ODP2) polypeptide comprises the amino acid sequence of any of SEQ ID NO: 18, 20, 63, 65, or 67, or wherein the BBM polypeptide or the Ovule Development Protein 2 (ODP2) polypeptide is encoded by the nucleotide sequence of any of SEQ ID NO: 17, 19, 21, 62, 64, 66, or 68. 
     
     
       26. The method of  claim 17 , wherein the WUS/WOX homeobox polypeptide comprises the amino acid sequence of any of SEQ ID NO: 4, 6, 8, 10, 12, 14, or 16, or wherein the WUS/WOX homeobox polypeptide is encoded by the nucleotide sequence of any of SEQ ID NO: 3, 5, 7, 9, 11, 13, or 15 and wherein the BBM polypeptide or the Ovule Development Protein 2 (ODP2) polypeptide comprises the amino acid sequence of any of SEQ ID NO: 18, 20, 63, 65, or 67, or wherein the BBM polypeptide or the Ovule Development Protein 2 (ODP2) polypeptide is encoded by the nucleotide sequence of any of SEQ ID NO: 17, 19, 21, 62, 64, 66, or 68. 
     
     
       27. The method of  claim 4 , wherein the nucleotide sequence encoding the WUS/WOX homeobox polypeptide and the nucleotide sequence encoding the BBM polypeptide or the ODP2 polypeptide is excised. 
     
     
       28. The method of  claim 12 , wherein the nucleotide sequence encoding the WUS/WOX homeobox polypeptide and the nucleotide sequence encoding the BBM polypeptide or the ODP2 polypeptide is excised. 
     
     
       29. The method of  claim 20 , wherein the nucleotide sequence encoding the WUS/WOX homeobox polypeptide and the nucleotide sequence encoding the BBM polypeptide or the ODP2 polypeptide is excised.

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